1. health and fitness
2. disease

American Journal of Microbiological Research, 2014, Vol. 2, No. 6, 217-223
Available online at http://pubs.sciepub.com/ajmr/2/6/8
© Science and Education Publishing
DOI:10.12691/ajmr-2-6-8
Molecular Characterization and in Silico Analysis of a
Novel Mutation in TEM-1 Beta-Lactamase Gene among
Pathogenic E. coli infecting a Sudanese Patient
Hisham N Altayb1,*, Nagwa M El Amin2, Maowia M. Mukhtar1, Mohamed Ahmed Salih3, Mohamed A M Siddig4
1
Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan
Department of microbiology, Faculty of medicine, university of Khartoum, Sudan
3
Head department of biotechnology, Biotechnology Park, Africa city of technology, Sudan
4
Botany department, Faculty of Science, University of Khartoum, Sudan
*Corresponding author: [email protected]
2
Received November 19, 2014; Revised December 10, 2014; Accepted December 14, 2014
Abstract The presence of ESBLs in many E. coli strains are of serious concern, since these organisms are the
most common cause of different human infections. In this study we isolate an E. coli bacterium with high hydrolytic
activity against cefotaxime. The ESBLs production was confirmed by phenotypic confirmatory test, while the
ESBLs genes were detected by polymerase chain reaction (PCR). This isolate was positive for TEM gene and
negative for CTX-M and SHV genes. DNA sequencing was done for TEM gene. The nucleotide sequences and
translated proteins were subjected to BLAST for sequences similarity and homology, BLASTp result revealed a
substitution of aspartic acid in TEM-1(gb: AFI61435.1) to Threonine at position 262. In Silico tools was used for
mutation analysis and prediction of secondary and tertiary structure of wild and mutant type genes. We conclude that
our mutant gene is completely different from the wild types TEM-1 gene, within phenotypic and genotypic levels.
So we conclude a novel mutant TEM gene with ESBLs activity is been detected in Sudan Phylogenetic tree revealed
that the possible source of our gene is Iran.
Keywords: E. coli, ESBLs, Novel TEM gene, Insilco analysis, Sudan
Cite This Article: Hisham N Altayb, Nagwa M El Amin, Maowia M. Mukhtar, Mohamed Ahmed Salih, and
Mohamed A M Siddig, “Molecular Characterization and in Silico Analysis of a Novel Mutation in TEM-1 BetaLactamase Gene among Pathogenic E. coli infecting a Sudanese Patient.” American Journal of Microbiological
Research, vol. 2, no. 6 (2014): 217-223. doi: 10.12691/ajmr-2-6-8.
1. Introduction
The production of penicillinases and other β-lactamases
is the principal mechanism of resistance to beta-lactam
antibiotics among members of the Enterobacteriaceae
family [1,2]. Before the CTX-M era, TEM-type extendedspectrum β-lactamases (TEM-ESBL) were the most
prevalent mechanism of resistance to β-lactam antibiotics
in Enterobacteriaceae. They emerged from the parental
penicillinases TEM-1 and TEM-2 [3]. The TEM-type
ESBLs are derivatives of TEM-1 and TEM-2. TEM-1 was
first reported in 1965 from an Escherichia coli isolate
from a patient in Athens, Greece, named Temoneira
(hence the designation TEM) [4]. This TEM-1 producing
E. coli hydrolyzed ampicillin, and within a few years after
its first isolation, its plasmid-mediated resistance had
spread over the world and into many different members of
the Enterobacteriaceae family, including Pseudomonas
aeruguinosa, Haemophilus influenzae and Neisseria
gonorrhoeae. Other β -lactamases were then discovered
which were closely related to TEM-1 and TEM-2, but
which had the ability to confer resistance to the extended-
spectrum cephalosporins [5,6]. Hence these new β lactamases were coined extended-spectrum β–lactamases
(ESBLs).
TEM-1 is able to hydrolyze ampicillin at a greater rate
than carbenicillin, oxacillin, or cephalothin, and has
negligible
activity
against
extended-spectrum
cephalosporins. It is inhibited by clavulanic acid. TEM-2
has the same hydrolytic profile as TEM-1, but differs from
TEM-1 by having a more active native promoter and by a
difference in isoelectric point (5.6 compared to 5.4).
TEM-13 also has a similar hydrolytic profile to TEM-1
and TEM-2 [7]. Well over 200 TEM-type β -lactamases
have been described, of which the majority are ESBLs.
Their isoelectric points range from 5.2 to 6.5. The amino
acid changes in comparison with TEM-1 or TEM-2 are
documented at http://www.lahey.org/Studies/temtable.asp.
Previously other studies were conducted to detect and
characterize TEM and other ESBLs genes in Sudan
[23,26]. But in this study we did DNA sequencing to
know TEM gene type, and also we aimed to identify novel
TEM genes types by using Insilco analysis tools.
2. Materials & Methods
American Journal of Microbiological Research
2.1. Bacterial Isolates
An E. coli bacterium isolate was recovered from urine
specimen of 45 years old female, from Gizera state that
located in the central of Sudan, at May 2013, during a
study to detect and characterize ESBLs genes in this area,
this organism was show high hydrolytic activity to
cefotaxime, and was positive for TEM gene and negative
for CTX-M and SHV genes by PCR. So was selected for
DNA sequencing. Chromogenic agar media (Liofilchem
Co. Italy) and standard biochemical tests [8], were used in
the isolation and identification of bacteria. E. coli ATCC
25922 and Klebsiella pneumoniae ATCC 700603 were
used as controls.
218
PCR mixture was subjected to initial denaturation step at
94°C for 5-min, followed by 30 cycles of denaturation at
94°C for 45 seconds, primer annealing at 57°C for 45
seconds, followed by a step of elongation at 72°C for 60
seconds, the final elongation was at 72°C for 5 min [12].
PCR products was analyzed by electrophoresis in a 2%
agarose gel in TBE 1X that contain 2.5 µl of (20mg/ml)
ethidium bromide at 100V for 40 min. Bands were
visualized under U.V transilluminater (Uvite –UK).
2.5. Sequencing of TEM Gene
DNA purification and standard sequencing was
performed for both strands of TEM genes by Macrogen
Company (Seoul, Korea).
2.2. Antibiotic Susceptibility Testing
ESBL screening and confirmation along with
antimicrobial susceptibility test was done according to the
Clinical Laboratory Standards Institute (CLSI) guidelines
[9] (CLSI, 2012). We used antibiotic disks containing
Ceftazidime (CAZ: 30 μg), Cefotaxime (CTX: 30 μg),
Cefepime (CTX: 30 μg), Amoxyclav (AMC: 30 μg),
Gentamicin (GM: 10 μg), Meropenem (MRP: 10 μg),
ofloxacin (OF: 5μg), Tetracycline (TE: 10 μg), Amikacin
(AK: 30μg), Chloramphnicol (C: 30μg) and Ciprofloxacin
(CIP: 5 μg). An inhibition zone size of ≤ 22 mm with
Ceftazidime, ≤ 27 mm with Cefotaxime and ≤ 18 mm with
Cefepime were considered as potential ESBL producer.
2.3. Phenotypic detection of ESBLs
The ESBLs phenotype of the clinical isolate was
confirmed by using ceftazidime (30μg), ceftazidime (30μg)
plus clavulanic acid (10μg), cefotaxime (30μg),
cefotaxime (30 μg) plus clavulanic acid (10μg) and
cefepime (30 μg), cefepime (30 μg) plus clavulanic acid
(10μg), (Liofilchem Co. Italy). Phenotypic detection of
ESBLs was defined by a difference of ≥ 5 mm between
the disk that contain clavulanic acid and that without
clavulanic acid [9,10].
2.4. Genotypic detection of ESBLs
The DNA was extracted by guanidine chloride method
as described by Alsadig et al., [11]. The PCR was carried
out using thermal cycler (CONVERGYS® td peltier
thermal cycle, Germany), the following primers (Metabion,
GERMANY) were used, for bla TEM 971bp; TEM-F 5 ’
TCG GGG AAA TGT GCG CG 3’ and TEM-R 5 ’ TGC
TTA ATC AGT GAG GCA CC 3’, for bla SHV 797 bp;
SHV-F 5-GGTTATGCGTTATATTCGCC-3, SHV-R 5TTAGCGTTGCCAGTGCTC-3 and for bla CTX-M 550
bp; CTX-M-F SCS 5-ATG TGC AGY ACC AGT AA-3,
CTX-M-R 5-CCG CRA TAT GRT TGG TGG TG-3, in a
total reaction volume of 25 μl (5µl Master mix of Maxime
RT premix kit (iNtRON BIOTECHNOLOGY, Seongnam,
Korea), 0.6 μl of forward primer, 0.6 μl of reverse primer,
2µl DNA and 16.8 µl deionized sterile water). Then the
2.6. Bioinformatics Analysis
The sequences chromatogram was viewed by FinchTV
program, (http://www.geospiza.com/Products/finchtv.shtml).
Then the nucleotides sequences of the TEM beta-lactamases
gene were searched for sequences similarity using nucleotide
BLAST (http: //blast.ncbi.nlm.nih.gov/Blast.cgi.) [13].
Highly similar sequences were retrieved from NCBI and
subjected to multiple sequence alignment using BioEdit
software
[14].
In
GeneMarkS
version
4.25
(http://exon.gatech.edu/genemark/genemarks.cgi),
the
gene sequences were translated into amino acid sequence
[15]. Protein sequences similarity were searched with
BLASTP (http://blast.ncbi.nlm.nih.gov/ Blast.cgi CMD
=Web&PAGE_TYPEBlastDocs),
highly
similar
sequences were achieved from NCBI and subjected to
multiple sequence alignment and evolutionary analysis
using BioEdit software. Phylogenetic tree of TEM betalactamase gene and their evolutionary relationship with
those obtained from database was done online by Clustal
W2 http://www.ebi.ac.uk/Tools/msa/clustalw2/) [16]. The
secondary structures of predicted amino acids were carried
out by phyre2 [17]. Chimera version 1.9 software was
used to predict the tertiary model of protein [18]. Mutation
analysis was done online by project hope software
(http://www.cmbi.ru.nl/hope/report/2064?10) [19], and
Expasy (http://web.expasy.org/compute_pi/) [20].
3. Results
3.1. Phenotypic Detection of ESBLs
After the initial CLSI screening tests, the isolate was
found resistant to cefotaxime, amoxyclav, tetracycline,
nalidixic acid, chloramphnicol and ciprofloxacin, and sensitive
to meropenem, cefepime, ceftazidime and amikacin, see
Table 1. Then ESBLs production was confirmed by
combination disc diffusion test, cefotaxime/cefotaxime
+clavulanic acid gave a positive result (showed a difference
of > 5 mm), while ceftazidime/ceftazidime+clavulanic and
cefepime/cefepime+clavulanic were negative (showed a
difference of < 5 mm), as presented in Figure 1.
Table 1. The results of screening tests using different antibiotics
Antibiotics inhibition zone diameter (mm)
Isolate
CAZ
CTX
NA
C
MRP
CPM
AMC
CIP
AK
TE
E. coli
30
12
8
8
30
34
0
10
20
0
Abbreviations: CAZ= ceftazidime, CTX= cefotaxime, NA= nalidixic acid, C= chloramphnicol, MRP= meropenem, CPM= cefepime, AMC=
amoxiclave, CIP= ciprofloxacin, AK= amikacin, TE= tetracycline, OF= ofloxacin.
OF
0
219
American Journal of Microbiological Research
Figure 1. Phenotypic confirmatory test with CAZ= ceftazidime, CAL=
ceftazidime+clavulanic
acid,
CTX=
cefotaxime,
CTL=
cefotaxime+clavulanic
acid,
CPM=
cefepime,
FEL=
cefepime+clavulanic acid, on Muller Hinton agar
Analysis of TEM gene sequences was done to
determine their relationship to other TEM gene sequences
available in Gen-Bank database using BLAST nucleotide
algorithm
(http://www.ncbi.nlm.nih.gov/).
BLAST
analysis of TEM gene (971-bp) showed 99% identity to
nucleotide sequence of TEM-1 of different bacterial
species, that obtained from Gen-Bank database; E. coli
TEM-1 from China gb: AFI61435.1|, S. enterica TEM-1
from Island gb|AB571794.1, TEM-1B from Malaysia
gb|HQ625489.1, L. adecarboxylata TEM-1 from China
gb|JF910132.1 and K. pneumoniae TEM-1 from China
gb|KF268357.1). Our mutant TEM gene was different by
an inserted G at position 880 and deleted G at position 931
see Figure 2B. This mutation resulted in a substitution of
amino acid Aspartic acid to Threonine (D-T) at position
262, when compared to TEM-1 gene (gb: AFI61435.1|)
and TEM-169 (gb|AFI61433.1|) as presented in Figure 3.
3.2. Genotypic Detection of ESBLs
3.4. Multiple Sequence Alignment
The selected isolate showed a band typical in size to
TEM beta-lactamase gene (971bp), see Figure 2A, and
was negative for both CTX-M (550bp) and SHV (797bp)
beta-lactamase gene.
The multiple sequence alignment of the mutant isolate
with similar nucleotide sequences that obtained from
BLASTn was carried out to find the homology and
evolutionary relation between these sequences. As shown
by BioEdit software there is an inserted and deleted G and
at very conserved region, see Figure 2C.
3.3. Bioinformatics Analysis
Figure 2. A. PCR amplification of TEM gene of the E. coli isolate on 2% agarose gel electrophoresis. Lane 1 DNA ladder: MW 100-1500bp. Lane 2
control negative. Lane, 3 control positive Lane 4 showing typical band size of (971bp) corresponding to the molecular size of TEM gene. B. Mutant
TEM gene sequence chromatogram, shown by FinchTV software. C. BioEdit multiple sequence alignment. The inserted and deleted G in Sudan isolate
illustrated by the arrows
3.5. Protein
Structure
Secondary
and
Tertiary
The prediction of secondary protein structure of wild
and mutant TEM genes revealed that the wild type contain
46% alpha helix, 14% Beta strand and 11% Disordered
regions, while the mutant gene contain 45%, Alpha helix,
15% Beta strand and 12% disordered regions as presented
in Figure 4 A1, A2. The prediction of 3D structure of the
wild and mutant TEM genes was done by Chimera
software version 1.9 as presented in Figure 4 B1, B2.
American Journal of Microbiological Research
220
Figure 3. Amino acid multiple sequence alignment of mutant TEM-Sudan beta-lactamase gene compared to other TEM gene from database, the mutant
Threonine (T) at position 262 shown by an arrow. The alignment was performed using the Clustal W2 sequence alignment
Figure 4. Left: wild type (A1, B1)of predicted amino acid Aspartic acid (ASP) at position 262 of TEM-1 gene (gb: AFI61435.1). Right: mutant type
(A2, B2) of predicted amino acid Threonine (THR) at position 262. A1, A2. The secondary protein structure of wild and mutant gene that drawn by
Phyre2 software. B1, B2. Tertiary protein structure of wild and mutant genes that drawn by Chimera software version 1.9
221
American Journal of Microbiological Research
3.6. Expasy and Project Hope
Table 2. A comparison of wild type and mutant type residues of
TEM genes
Feature
Wild residue
Mutant residue
Size
Normal
Smaller than wild-type
residue
Charge
Negatively charged
Neutral
Hydrophobicityvalue
Hydrophobic
More hydrophobic
Structure
Located in its preferred
secondary structure
Prefers to be in another
secondary structure
The theoretical isoelectric point of wild type gene was
pI/Mw: 5.32 / 29499.79 and for mutant type was pI/Mw:
5.44 / 29485.81. Project Hope software revealed many
differences between wild and mutant residues as shown in
Table 2.
3.7. Phylogenetic Tree
The drawn phylogenetic tree revealed that our mutant
TEM gene is so closed to Iranian TEM-1 and TEM-169,
see Figure 5.
Figure 5. Phylogenetic tree of mutant Sudan TEM beta-lactamase gene and other TEM genes that obtained from database
4. Discussions
The reports of novel blaTEM enzymes that have both
an extended spectrum of activity and resistance to betalactamase inhibitors suggest that the TEM-type enzymes
are continuing to evolve in the face of current therapies
[21,22]. The rapid identification of an ESBL gene would
result in the initiation of the appropriate therapeutic
intervention sooner and may result in fewer clinical
failures. In this study we isolate an E. coli bacterium that
produce TEM gene, from a Sudanese patient, like many
other studies detect and characterize ESBLs genes in
Sudan [23,24,25,26]. This isolate showed a high
hydrolytic activity against cefotaxime than ceftazidime
even its negative for CTX-M gene, this agree with that
some TEM genes have an enhanced activity against
cefotaxime like TEM-3 [27]. In addition to cefotaxime
also this isolate resistant to ciprofloxacin, tetracycline,
amoxyclav, ofloxacin chloramphnicol and nalidixic acid,
this may indicate that the plasmid that carry the ESBL
gene may carry other resistant genes, like for quinolones
and aminoglycoside [28].
The BLASTn searching and BioEdit multiple sequence
alignment revealed an inserted G at position 880 and
deleted G at position 931, at very conserved region. The
translation of amino acid sequences revealed a substitution
of Aspartic acid in TEM-1 (gb: AFI61435.1) from Iran to
Threonine in our mutant TEM gene at position 262. This
mutation may be the cause of ESBL activity, because this
mutation located at beta-lactamase/transpeptidase-like
domain which is the penicillin-binding domain of PBPs,
that function as DD-transpeptidases, its catalyze the final
step of cell wall biosynthesis by cross-linking two strands
of peptidoglycan, or DD-peptidases [29]. This agrees with
that many ESBLs TEM genes evolved from one or more
amino acid substitution around the active site [30]. This
mutation increased protein isoelectric point from pI; 5.32
to pI; 5.44, this like in TEM-2 the first derivative of TEM1, had a single amino acid substitution from the original βlactamase, that is cause a shift in the isoelectric point from
a pI of 5.4 to 5.6 [31].
The secondary and tertiary structure of wild and mutant
type proteins also changed, the percentage of alpha and
beta helixes was changed in secondary protein structure,
the shape and ionic interaction also changed in tertiary
protein structure. These changes may affect protein
functions and folds. The affection of insertions,
duplications, and deletions of sequence contribute to the
structural and functional diversity of proteins has been
relatively well studied in model proteins [32]. The mutant
residue was found completely different from the wild type
residue; in 3D-structure wild-type residue is located in its
preferred secondary structure, while mutant residue
prefers to be in another secondary structure, therefore the
local conformation will be slightly destabilized. The wildtype residue is not conserved at this position, while the
mutant residue was not among the residue types observed
at this position in other, homologous sequences which
might indicate that the mutation is possibly damaging to
the protein because the mutation occurred near a highly
conserved position. There is a difference in charge
between the wild-type and mutant amino acid, the charge
of the wild-type residue is lost by this mutation, this can
cause loss of interactions with other molecules. The wildtype and mutant amino acids differ in size, the mutant
residue is smaller than the wild-type residue, and this will
American Journal of Microbiological Research
cause a possible loss of external interactions. The
hydrophobicity of the wild-type and mutant residue also
different. The phylogenetic tree revealed that the possible
source of our new gene is Iran, this may be due that Iran is
the nearest country to Sudan among these countries.
5. Conclusions
We conclude that a novel mutant TEM gene with
ESBLs activity is been detected in Sudan, this is new gene
is completely different from TEM-1 gene in its phonotypic
and genotypic characterization. ESBLs genes are rapidly
evolved among pathogenic bacteria, thus study like this to
detect a new antibiotic resistant gene variants could guide
the choice of optimal antibiotic therapy for successful
treatment, thus improving the outcomes for patients with
severe bacterial infections.
Acknowledgments
The authors kindly thank the staff and students of
institute of endemic diseases, university of Khartoum,
Sudan. We acknowledge Ms. Haifa from Wad Madani
Hospital and Mr. Riham from Haj Alsafi Hospital for their
support in sample collection. And also we acknowledge
Ms. Suhair Ramadan from Sudan University of science
and technology for her support.
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[20]
Competing Interests
The authors declare that they have no conflict of
interests.
[21]
[22]
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